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Background Inflammatory bowel diseases (IBDs) involve an aberrant host response to intestinal microbiota causing mucosal inflammation and gastrointestinal symptoms. Patient-reported outcomes (PROs) are increasingly important in clinical care and research. Our aim was to examine associations between PROs and fecal microbiota in patients 0 to 22 years of age with IBD. Methods A longitudinal, prospective, single-center study tested for associations between microbial community composition via shotgun metagenomics and PROs including stool frequency and rectal bleeding in ulcerative colitis (UC) and abdominal pain and stool frequency in Crohn’s disease (CD). Mucosal inflammation was assessed with fecal calprotectin. A negative binomial mixed-effects model including clinical characteristics and fecal calprotectin tested for differentially abundant species and metabolic pathways by PROs. Results In 70 CD patients with 244 stool samples, abdominal pain correlated with increased relative abundance of Haemophilus and reduced Clostridium spp. There were no differences relative to calprotectin level. In 23 UC patients with 76 samples, both rectal bleeding and increased stool frequency correlated with increased Klebsiella and reduced Bacteroides spp. Conversely, UC patients with lower calprotectin had reduced Klebsiella. Both UC and CD patients with active symptoms exhibited less longitudinal microbial community stability. No differences in metabolic pathways were observed in CD. Increased sulfoglycolysis and ornithine biosynthesis correlated with symptomatic UC. Conclusions Microbial community composition correlated with PROs in both CD and UC. Metabolic pathways differed relative to PROs in UC, but not CD. Data suggest that microbiota may contribute to patient symptoms in IBD, in addition to effects of mucosal inflammation.

Author(s): Denson, Lee A; Jurickova, Ingrid; Karns, Rebekah; Shaw, Kelly A; Cutler, David J; Okou, David; Valencia, C Alexander; Dodd, Anne; Mondal, Kajari; Aronow, Bruce J; Haberman, Yael; Linn, Aaron; Price, Adam; Bezold, Ramona; Lake, Kathleen; Jackson, Kimberly; Walters, Thomas D; Griffiths, Anne; Baldassano, Robert N; Noe, Joshua D; Hyams, Jeffrey S; Crandall, Wallace V; Kirschner, Barbara S; Heyman, Melvin B; Snapper, Scott; Guthery, Stephen L; Dubinsky, Marla C; Leleiko, Neal S; Otley, Anthony R; Xavier, Ramnik J; Stevens, Christine; Daly, Mark J; Zwick, Michael E; Kugathasan, Subra | Abstract: BACKGROUND:Granulocyte-macrophage colony-stimulating factor auto-antibodies (GMAbs) suppress neutrophil-extrinsic GM-CSF signaling and increase risk for stricturing behavior in Crohn's disease (CD). We aimed to define clinical, genomic, and functional associations with neutrophil-intrinsic GM-CSF signaling. METHODS:Missense mutations in CSF2RA, CSF2RB, JAK2, STAT5A, and STAT5B were identified using whole-exome sequencing in 543 pediatric inflammatory bowel disease (IBD) patients. Neutrophil-intrinsic GM-CSF signaling was defined using the GM-CSF-induced STAT5 stimulation index (GMSI) in 180 pediatric IBD patients and 26 non-IBD controls. Reduced GM-CSF signaling (GMSI-Lo) was defined as the 20th percentile within the control group. Variation in neutrophil phospho-protein abundance, bacterial killing, and the global pattern of gene expression with the GMSI was determined. RESULTS:We validated 18 potentially damaging missense mutations in CSF2RA and CSF2RB. CSF2RA A17G carriage increased from 10% in those with intact neutrophil GMSI to 32% in those with low GMSI (P = 0.02). The frequency of reduced Staphylococcus aureus killing increased from 17% in those with intact neutrophil GMSI to 35% in GMSI-Lo neutrophils (P = 0.043). Crohn's disease neutrophils with low GMSI exhibited specific alterations in phospho-protein networks and genes regulating cytokine production, wound healing, and cell survival and proliferation. Stricturing behavior increased from 7% in patients with both low GMAb and intact GMSI to 64% in patients with both elevated GMAb and low GMSI (P l 0.0001). CONCLUSIONS:Low/normal neutrophil-intrinsic GM-CSF signaling is associated with CSF2RA missense mutations, alterations in gene expression networks, and higher rates of disease complications in pediatric CD.

Objectives Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are characterized by an aberrant host response to intestinal microbiota causing mucosal inflammation and gastrointestinal symptoms. Patient reported outcome measures (PROs) are increasingly utilized in clinical care and research. Our study aim was to determine if fecal microbial shifts were associated with PROs in children and young adults with IBD. Methods A longitudinal prospective single center study of 93 patients tested for association between fecal microbial shifts, mucosal inflammation as measured by fecal calprotectin, and self-reported symptoms including diarrhea, rectal bleeding, and abdominal pain. For CD, abdominal pain and diarrhea determined disease activity or overall PRO. For UC, diarrhea and rectal bleeding were used. Fecal calprotectin and shotgun metagenomic sequencing were performed on all samples. Demographic and clinical characteristics (Table 1) were incorporated into a negative binomial mixed-effects model in “R” to identify differentially abundant species by PRO and fecal calprotectin. Metabolic pathways were mapped using the HUMAnN2 pipeline and compared to overall PRO, individual symptoms and fecal calprotectin level. Results In 70 CD patients with 244 stool samples, there was no association between symptoms and fecal calprotectin. In 23 UC patients with 76 stool samples, increased fecal calprotectin was associated with rectal bleeding (OR 4.93 [1.18, 20.64], p=0.03). Examination of differentially abundant species in those with self-reported active UC showed increased Klebsiella species and reduced Bacteroides. Conversely, UC patients with fecal calprotectin < 100 µg/gm had reduction in Klebsiella and increase in Bifidobacteria and Bacteroides (Figure 1). Analysis of differentially abundant species in those with abdominal pain in CD showed increase in Haemophilus and reduction in Bacteroides. No microbial shifts were identified in CD patients in association with overall PRO, diarrhea, nor with fecal calprotectin < 250 µg/gm. Metabolic pathway analysis showed no differences in those with CD. In UC patients, increases in sulfoglycolysis and ornithine biosynthesis were associated with overall PROs. Conclusions Fecal microbial shifts including decreased commensals such as Bacteroides correlate with UC patient symptoms. Increased fecal calprotectin level was associated with rectal bleeding in these patients, but not diarrhea. In CD, there was no association with fecal calprotectin and symptoms, and microbial shifts were detected in association with abdominal pain. Similarly, metabolic pathways differed relative to patient-reported symptoms in UC, but not in CD. Data suggests that microbial shifts may directly contribute to symptoms in children and young adults with IBD.

Background Clostridioides difficile infection and colonization are common in pediatric Crohn’s disease (CD). Our aims were to test the relationship between C. difficile positivity and bowel resection surgery and to characterize microbial shifts associated with C. difficile carriage and surgery. Methods A retrospective single-center study of 75 pediatric CD patients tested for association between C. difficile carriage and bowel resection surgery. A prospective single-center study of 70 CD patients utilized C. difficile testing and shotgun metagenomic sequencing of fecal samples to define microbiota variation stratified by C. difficile carriage or history of surgery. Results The rate of bowel resection surgery increased from 21% in those without C. difficile to 67% in those with (P = 0.003). From a Kaplan-Meier survival model, the hazard ratio for time to first surgery was 4.4 (95% CI, 1.2–16.2; P = 0.00) in patients with positive C. difficile testing in the first year after diagnosis. Multivariable logistic regression analysis confirmed this association (odds ratio 16.2; 95% CI, 2.2–120; P = 0.006). Larger differences in microbial abundance and metabolic pathways were observed in patients with prior surgery than in those with C. difficile carriage. Depletion of Alistipes and Ruminococcus species and reduction in methionine biosynthesis were noted in patients with both C. difficile carriage and past surgery. Conclusions A positive C. difficile test during the first year after diagnosis is associated with decreased time to first bowel resection surgery in pediatric Crohn’s disease. Depletion of beneficial commensals and methionine biosynthesis in patients with C. difficile carriage may contribute to increased risk for surgery.